RNA after sorting (yet again!)
utk at urieltk.com
Wed Sep 17 18:38:02 EDT 2008
I am sorting human monocyte derived DCs on our beloved ARIA for RNA for microarrays. I tried obtaining RNA from a "regular" sort, and the yield and purity were excellent but the RNA was degraded, not a lot, but too much for microarrays. I use quiagen's RNEasy kits, so I cannot sort directly into lysis buffer. I'm going to do an "RNA sort", and I have some of questions that seemingly haven't yet been asked.
1) Has anyone used PBS prepared with DEPC treated water instead of regular PBS? I think that is a bit too paranoid, but since it was suggested to me... I strongly suspect the degradation problem is mainly from the cells themselves sitting on ice, dying and releasing RNAses, + FBS, so DEPC water wouldn't make much of a difference I think, but maybe I'm wrong. I do seem to recall that DEPC treated water is toxic to cells though. Can anyone confirm? Has anyone tried this approach?
2) Since I've been sorting into FBS, and F-B-SERUM has many RNAses (or so I've been told), I'm changing it into BSA. Of course, it's B-SERUM-A, so RNAses probably still lurk, but probably less so as well. Any comments or experience in this respect? Will BSA "cushion" the cells as well as FBS?
3) QUiagen has recently introduced a product they call RNAprotect, which is a relative of RNAlater but specifically brewed for cultured and sorted cells (yes! someone created a buffer specially for sorting!). They recommend a 5:1 ratio of RNAprotect to cell medium, like RNAlater. Did anyone who used RNA later tried with a lower ratio? did it work OK? I'm using 15 mL tubes and I'd have to switch them very quickly (and expend a lot of the RNA protect reagent).
4) In that regard, given that most of the sorted drops is actually sheath fluid, what would be the strategy to get a)the smallest drops and b)the fewest drops sorted per event, so that the tubes fill as slowly as possible?
As a comment on the ARIA I've mentioned before on the list, I am sure BD had a black magic wizard in the ARIA design team. A week ago we spent 6 hours of grueling hell, changing every possible element and trying every possible fix, to end up throwing away millions of precious DCs unsorted and a couple hundred thousand sorted fellows who just waited too long for the friends that never came. This Monday, we sorted millions and millions of DCs for more than 5 hours, and except restarting the stream once, there wasn't a single incident! Back to back sorting, from the 1st cell to the last one. I knew praying to the god(s) wasn't working. Black magic I tell you! THAT is the answer.
Uriel Trahtemberg, M.D. M.Sc.
The Laboratory for Cellular and Molecular Immunology
The Hebrew University - Hadassah Medical Organization
Jerusalem - ISRAEL
"Life is a shipwreck in which, at the last moment, only the ship survives"
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